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ALKA VITA - ALKA HYDROXY
IN-VIVO MELANOMA CANCER
Note: Alka Vita/AlkaHydroxy names used only for testing
Product name: Modified Liquid Silicate
New marketing name:
MELANOMA CANCER TEST
Efficacy Evaluation of Antitumor Activity
of Alka Vita - Alkahydroxy
in the LOX-GFP Human Melanoma Model
Final Report by:
Anti-Cancer Lab – San Diego California
March 4, 2005
Efficacy Evaluation of Antitumor Activity of Alkahydroxy in the LOX-GFP Human Melanoma Model
1.0 OBJECTIVE The purpose of this study was to evaluate the antitumor activity of Alkahydroxy on
tumor growth in LOX-GFP human melanoma model through intradermal injection of LOX cells in nude mice.
2.0 INVESTIGATORS Sponsor: Cisne Enterprises Inc. Sponsor Investigators: Michael D Radcliffe AntiCancer, Inc.: Yunling Jing, M.D., Principal Investigator Zhijian Yang, Study Director Shigeo Yagi, Ph.D., General Manager Robert M. Hoffman, Ph.D., President 3.0 STUDY PERIOD This study was conducted between January 3, 2005 and February 18,2005. 4.0 MATERIALS AND METHODS Animals: Male NCr nude mice between 5 and 6 weeks of age were used in this study.
The animals were bred and maintained in a HEPA filtered environment with cages, food and bedding sterilized by autoclaving. The breeding pairs were obtained from Charles River Laboratories (Wilmington, MA). The animal diets were obtained from Harlan Teklad (Madison, WI). 0.008% (w/v) Ampicillin (Sigma, St Louis, MO) was added to the autoclaved drinking water. A total of 15 animals were used for this study.
Study drugs: Alkahydroxy A and Alkyhydroxy B were supplied by Cisne Enterprises Inc. (see protocol).
GFP expression vector. The pLEIN vector was purchased from Clontech (Palo Alto,
CA). The vector expresses enhanced GFP and the neomycin resistance gene on the same
bicistronic message that contains an internal ribosome entry site.
Cell culture, vector production, transfection, and subcloning. PT67, an NIH3T3-
derived packaging cell line, expressing the 10 A1 viral envelope, was purchased from
Clontech. PT67 cells were cultured in DMEM supplemented with 10% fetal bovine
serum. For vector production, packaging cells (PT67), at 70% confluence, were incubated
with a precipitated mixture of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-
trimethylammoniummethyl
sulfate reagent and saturating amounts of pLEIN plasmid for 18h. Fresh medium was
replenished at this time. The cells were examined by fluorecence microscopy 48h post-
transfection. For selection, the cells were cultured in the presence of 200-1000 g/ml
G418 for 7 days.
Retroviral GFP Transduction of LOX Cells. For GFP gene transduction, LOX cells
(National Cancer Institute, Bethesda, MD) at 25 % confluence were incubated with a 1:1
precipitated mixture of retroviral supernatants of PT67 cells and RPMI 1640 (Life
Technologies, Inc.) containing 10 % fetal bovine serum (Gemini Bioproducts) for 72 h.
Fresh medium was replenished at this time. Cells were harvested by trypsin-EDTA 72 h
after transduction and subcultured at a ratio of 1:15 into selective medium that contained
200 g/kg G418. The level of G418 was increased stepwise to 800 g/kg for LOX cells.
Clones expressing GFP were isolated with cloning cylinders (Bel-Art Products,
Pequannock, NJ) using trypsin-EDTA and then amplified and transferred by conventional
culture methods.
Doubling Time of Stable GFP Clones. GFP or nontransduced cells were seeded at 1.5 x
104
in 35-mm culture dishes. The cells were harvested and counted every 24 h using a
hemocytometer (Reichert Scientific Instruments, Buffalo, NY). The doubling time was
calculated from the cell growth curve over 6 days.
Intradermal Injection of LOX. Fifteen 6-week-old male NCr mice were injected
intradermally with a single dose of 1 x 106
LOX-GFP cells. Cells were first harvested by
trypsinization and washed three times with cold serum-free medium and then injected in a
total volume of 0.1 ml within 30 min of harvesting. Cells were inoculated into dorsal skin
using a 30 G1/2 precision glide needle (Becton Dickinson) and a 1-ml latex-free syringe
(Becton Dickinson).
Whole-body optical imaging of green fluorescent protein-expressing tumors and
metastases. A Leica stereo fluorescence microscope model LZ2 equipped with a mercury
Group & Agent Schedule Route of
administration
Number of
Mice
1, Untreated control - - 5
2, Alkahydroxy A Three times a day for
6 weeks Topical 5
3, Alkahydroxy B Three times a day for
6 weeks Topical 5
lamp power supply was used. Selective excitation of GFP was produced through a D425/60 band-pass filter and 470 DCXR dichroic mirror. Emitted fluorescence was collected through a long-pass filter GG475 (Chroma Technology, Brattleboro, VT) on a Hamamatsu C5810 3-chip cooled color CCD camera (Hamamatsu Photonics Systems, Bridgewater, NJ). Images were processed for contrast and brightness and analyzed with the help of Image Pro Plus 3.1 software (Media Cybernetics, Silver Spring, Maryland). High-resolution images were captured directly on the computer or continuously through video output on a high -resolution Sony VCR. Analysis of metastases: Metastases were detected by direct GFP open imaging at necropsy. Locations of metastases were recorded for each test animal. Study design: The intradermal (orthotopically) injected animals used for the study were divided into 3 groups 4 days after surgery. Groups for each of the cohort conditions were randomly chosed. Treatment began 4 days after implantation three times a day and lasted for six weeks (The concentration of Alkahydroxy B was increased to 25% on January 25th, 2005, lasting 10 days; and the concentration of Alkahydroxy B was increased again to 50% on February 4th, 2005, lasting two weeks). Table 1 shows the study design and compounds used in each group. Table 1 Treatment protocol Study endpoint: The experiment was terminated 47 days after tumor cell injection (6 weeks treatment) due to the poor health status of the test animals. Data collection:
GFP Imaging - GFP whole body images for each mouse were obtained once a
week after initial treatment.
Tumor Measurement - Tumor measurements were determined by GFP whole
body imaging.
Body weights - Body weight for each animal was measured once a week after
initial treatment. An electronic balance was used for body weight measurement.
Final tumor weights - The final primary tumor weight for each animal were
determined with an electronic balance.
GFP open imaging – At the end of the study, GFP open imaging was conducted.
The primary tumor and major metastatic organs were explored under fluorescence
microscopy at necropsy.
Statistical methods used in efficacy evaluation: Final primary tumor weights in each
group were analyzed using the ANOVA test (with the Dunnett’s two sided test) with an = 0.05. Tumor metastatic rates of all groups were analyzed with Fisher’s exact test with
an = 0.05.
5.0 RESULTS
Efficacy on primary tumor: At the end of the study, the final primary tumor weights in
treated groups were compared to that of the untreated control group using the Dunnett’s
two-sided test. Statistical significant differences were obtained in Alkahydroxy A treated
group (p<0.05), verses the untreated control group. The results are shown in Table 2.
Table 2. Efficacy of Alkahydroxy A & B on final primary tumor weight
Group Mean final tumor
weight (g)) P-value
*
1, Untreated control 8.1 -
2, Alkahydroxy A 3.9 0.034
3, Alkyhydroxy B 4.7 0.091
* All treated groups are versus the untreated control group with Dunnett’s two-sided test.
Efficacy on metastasis rate: At the end of the study, all animals were opened and imaged to
examine metastasis. Metastatic incidences in different organs were determined
Group # of tested animals Superficial axillary L.N.
MIa Pb
1 Untreated control 5 0 -
2 Alkahydroxy A 5 1 1.000
3 Alkyhydroxy B 5 0 1.000
by direct GFP open imaging. The metastases and p-value for each organ in treated groups were compared to that of the untreated control group by using Fisher’s exact test (Table 3). No statistical significant difference was seen in either treatment compared to the untreated control. The results are shown in Table 3. Table 3 Efficacy on Metastatic rate
a MI Metastatic Incidence. b All treated groups compared to the untreated control group by Fisher’s exact test.
Body weight: No acute loss of body weight within the duration of the experiment was observed (see “Body weight graph”). 6.0 CONCLUSION The results showed that Alkahydroxy A significantly inhibited the growth of primary tumor (p<0.05), verses the untreated control group. No significant difference was seen regarding metastases for both treated groups compared with the untreated control group. There was no evidence of toxicity as shown in the body weight graph.
Yunling Jing, M.D., Principal Investigator Date:
Zhijian Yang, M.D., Study Director Date: Signatures on Original
Shigeo Yagi, Ph.D., General Manager Date:
Robert M. Hoffman, Ph.D., President Date:
8.0 REFRENCES Hoffman, R. M., Orthotopic metastatic mouse model for anticancer drug discovery evaluation: a bridge to the clinic. Investigational New Drugs, 17: 497-500, 1999. Yang, M., et al. Whole-body optical imaging of green fluorescent protein-expressing tumors and metastasis. Proc. Natl. Acad. Sci. USA 97, 1206-1211, 2000. Yang M., et al. Genetically fluorescent melanoma bone and organ metastasis models. Clinical Cancer Research. 5 3549-3559, 1999.
• DATA (TABLES, GRAPHS AND PHOTOS)
222426283032343638404244
1 8 15 22 28 36 42
1/7/2005 1/14/2005 1/21/2005 1/28/2005 2/3/2005 2/11/2005 2/17/2005
Mean
Bo
dy W
eig
ht
(g)
Days & Date of Initial Treatment
Efficacy Evaluation of Antitumor Activity of Alkahydroxy in the LOX-GFP Human Melanoma Model
Mean Body Weight Graph
Untreated control
Alkahydroxy A
Alkahydroxy B
0
200
400
600
800
1000
1200
1 8 15 22 28 36 42
1/7/2005 1/14/20051/21/20051/28/2005 2/3/2005 2/11/20052/17/2005
Me
an
Tu
mo
r A
rea
(m
m2)
Days & Date of Initial Treatment
Efficacy Evaluation of Antitumor Activity of Alkahydroxy in the LOX-GFP Human Melanoma Model
Mean Tumor Area Graph
Untreated control
Alkahydroxy A
Alkahydroxy B
0
20
40
60
80
100
120
Untreated control Alkahydroxy A Alkahydroxy B
Me
an
Tu
mo
r A
rea
(m
m2)
Groups
Efficacy Evaluation of Antitumor Activity of Alkahydroxy in the LOX-GFP Human Melanoma Model
Mean Tumor Area Graph from FOTI (1-21-05)
p=0.118 p=0.196
0
50
100
150
200
250
300
350
400
450
Untreated control Alkahydroxy A Alkahydroxy B
Me
an
Tu
mo
r A
rea
(m
m2)
Groups
Efficacy Evaluation of Antitumor Activity of Alkahydroxy in the LOX-GFP Human Melanoma Model
Mean Tumor Area Graph from FOTI (2-3-05)
p=0.426 p=0.235
0100200300400500600700800900
Untreatedcontrol
Alkahydroxy A Alkahydroxy BMe
an
Tu
mo
r A
rea
(m
m2)
Groups
Efficacy Evaluation of Antitumor Activity of Alkahydroxy in the LOX-GFP Human Melanoma
Model
Mean Tumor Area Graph from FOTI (2-17-05)
p=0.257 p=0.19
0
2
4
6
8
10
12
Untreated control Alkahydroxy A Alkahydroxy B
Me
an
Tu
mo
r W
eig
ht
(g)
Groups
Efficacy Evaluation of Antitumor Activity of Alkahydroxy in the LOX-GFP Human Melanoma
Model
Mean Tumor Weight Graph (2-18-05)
p*=0.034
* Both treated groups were compared to the untreated control
with Dunnett's two sided test.
p*=0.091
Efficacy Evaluation of Antitumor Activity of Alkahydroxy in the LOX-GFP Human Melanoma Model GFP Open Images (2/18/05)
Untreated control, #3
Alkahydroxy B, #5
Alkahydroxy A, #1